Parvovirus B19 (B19) is a small ssDNA-containing virus that has evolved to be restricted in its replication to erythroid progenitor cells in the human bone marrow. B19 binds to erythroid cells through blood group P antigen or globoside (10). However, it has been demonstrated that P antigen alone is not sufficient for parvovirus B19 infection (54) and that functionally activated α5β1 integrin serves as a coreceptor for internalization into human cells (53). Subsequently, the Ku80 subunit of the DNA double-strand break repair protein Ku, which also functions as an adhesion receptor for fibronectin (31), was reported to also provide coreceptor activity (34).
The human epidermal growth factor receptor 2 (HER2/neu) has been reported to be overexpressed on ˜25% of human breast cancers by up to 100-fold the levels present on non-cancerous cells and on ˜30-40% of medulloblastomas by up to 5-fold (3, 11, 47). HER2-overexpression has been documented to result in constitutive activation of kinase signaling (57). HER2/neu-positive breast cancers are characterized by poor differentiation, high rates of proliferation, lymph node involvement, a relative resistance to certain types of chemotherapy and poor prognosis (11). About 30% of breast cancer patients present with bone marrow metastases at diagnosis (9). HER2/neu expression is seen in micrometastatic tumors in the bone marrow as well as in primary tumor cells and detection of HER2/neu in circulating tumor cells suggests a stable HER2/neu expression during metastatic spread of breast cancer (21, 35, 39, 50). Thus targeting HER2/neu on breast cancer cells is an attractive therapeutic approach and a humanized anti-HER2 monoclonal antibody (h4D5, trastazumab, Herceptin®) has been developed and tested in clinical trials (17, 25, 40, 42) (38). The mechanisms of Herceptin® action are thought to involve antibody-mediated cytotoxicity (5), blocking of receptor dimerization, removal of HER2/neu from the cell surface by endocytosis, and inhibition of HER2/neu extracellular domain (ECD) shedding by blocking a proteolytic cleavage site, which prevents constitutive tyrosine kinase activation of ECD-less receptors (13, 30) (23, 26, 48). When used as single agent in patients with HER2/neu-positive tumors, however, Herceptin® treatment had relatively high relapse rates (36) and it is currently used with combination chemotherapy (38).
The complementarity-determining regions (CDRs) of antibodies mediate their high-affinity binding and specificity to antigens (4) and peptide analogs of CDRs have been developed for antibodies with known sequences and structures (43). The HER2/neu-binding peptidomimetic (AHNP) was derived from the structure of the CDR-H3 loop of the anti-HER2 antibody rhu 4D5, and was shown to bind to cell surface-expressed HER2/neu, albeit with lower affinity than whole antibodies, and to inhibit HER2/neu kinase activity, modestly down-modulate HER2/neu and sensitize tumor cells to apoptosis when used in conjunction with ionizing radiation or chemotherapeutic agents (7, 37) (28).
Integrins have been suggested to play a cooperative role during oncogenesis, partly attributed to their reciprocal signaling to growth factor receptors (1) (20, 32, 51). Targeted disruption of the β1 integrin chain in a transgenic mouse model of human breast cancer demonstrated a critical role of β1 integrin in initiation and maintenance of mammary tumor growth in vivo (56) and co-clustering of HER2/neu and integrin β1 was demonstrated to be critical for induction of cell migration and survival during breast cancer progression (52). Integrin β1 is known to play an important role in hematopoietic stem and progenitor cell interaction with the bone marrow microenvironment (44), and has recently been identified as one of the adhesion receptors involved in cancer cell survival during genotoxic stresses induced by ionizing radiation and cytotoxic drugs (14, 15, 46).
Equipping viral vectors with the capability to target both HER2/neu and β1 integrins, could allow the delivery of a cytotoxic insult to HER2/neu-positive cancer cells in microenvironments, such as the bone marrow, where prosurvival signals mediated through β1 integrins prevent tumor cell eradication with conventional radiation and chemotherapies. We report here the introduction of the AHNP peptide into the capsid of B19 vectors to replace the endogenous P antigen binding site and demonstrate retargeting of B19 vectors to HER2/neu-positive tumor cells.